Application of SNAPIN protein and its encoding gene in anti-influenza virus

Abstract

The present invention discloses a host protein SNAPIN. Through in vitro research, it is found that the SNAPIN protein has the effect of antagonizing influenza virus replication, and the SNAPIN protein has the effect of enhancing the expression of interferon and interferon-induced genes, and has application value in the preparation of anti-influenza virus drugs or immune enhancers.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to the application of a SNAPIN protein and its encoding gene in anti-influenza virus. Background Art

[0002] Influenza A virus (IAV) can infect humans and animals, seriously threatening the development of the aquaculture industry and public health safety, and posing a potential threat to human health. Currently, various subtypes of influenza viruses such as H5N1, H6N1, H7N7, H7N9, H9N2, and H10N8 cross the host barrier and infect humans from time to time, and this phenomenon is the result of the interaction between the virus and the host.

[0003] Currently, the main anti-influenza virus drugs include amantadine, rimantadine, oseltamivir, interferon, etc. However, during long-term use of the drugs, drug-resistant strains are likely to be produced or the virus may develop immune escape. If some drugs are used improperly, serious adverse reactions such as acute renal failure may occur.

[0004] IAV belongs to the family Orthomyxoviridae and the genus Influenzavirus. Its genome is eight segments of single-stranded negative-strand RNA. There are two main glycoproteins on the surface of influenza virus particles, namely hemagglutinin (HA) and neuraminidase (NA). According to the different antigenicity of HA and NA, IAV can be divided into 18 HA subtypes and 11 NA subtypes. The internal genes of the virus are PB2, PB1, PA, NP, M, and NS, which encode different proteins respectively. Inside the viral lipid bilayer is the matrix protein M1, and inside the virus particle is the ribonucleoprotein complex (vRNP).

[0005] The SNAPIN protein (synaptosome-associated protein) is expressed in organs such as the brain, heart, liver, spleen, kidney, lung, and testis, primarily distributed in the cytoplasm, Golgi apparatus, lysosomes, and nucleus. The SNAPIN protein contains a 20-amino acid hydrophobic region at its N-terminus and two α-helical regions at its C-terminus, forming a coiled structure. The hydrophobic region and the α-helices are linked by a short peptide. The SNAPIN coiled structure typically interacts with other proteins, thereby exerting its biological functions. SNAPIN proteins play important roles in vesicle trafficking, autophagy, and regulation of viral replication. Overexpression of SNAPIN in human astroglioma cells (U373 cells) reduced UL70 nuclear entry and viral titers. However, knockdown of SNAPIN by siRNA increased UL70 nuclear entry and enhanced viral replication. These results suggest that SNAPIN regulates the cellular localization of HCMV UL70, thereby regulating viral DNA synthesis and progeny virion production. SNAPIN plays a crucial role in PRRSV replication. siRNA interference with SNAPIN inhibits viral replication, likely due to its interaction with GP5 and M proteins. Porcine hemagglutinating encephalomyelitis virus (PHEV) regulates viral replication through interaction between its S1 protein and SNAPIN. Downregulation of SNAPIN in PHEV-infected neurons inhibits viral replication, reduces infectivity, and leads to abnormal lysosomal accumulation during PHEV-induced autophagy.

[0006] Influenza viruses require the participation of host factors when replicating in the host. Currently discovered host factors can play two roles. First, some host factors can promote influenza virus replication, and second, some host factors can inhibit influenza virus replication. Drug design based on the theoretical basis that host factors inhibit influenza virus replication is called host-directed therapy. These host factors that inhibit influenza virus replication can serve as targets for the design of antiviral drugs and have potential application value. In current research, the development of host-directed antiviral drugs is still in its early stages. Designing drugs based on host-expressed proteins has many advantages, such as the difficulty in generating drug-resistant strains, no obvious toxic side effects, and the possibility of broad-spectrum antiviral effects.

[0007] Currently, there is no report on the role of SNAPIN in influenza virus replication. In this study, through 4D proteomics technology, it was screened that the expression of SNAPIN was down-regulated after A549 cells were infected with IAV. A lentiviral plasmid stably expressing SNAPIN protein was constructed using the pLVX-3×Flag vector and named pLVX-SNAPIN, and the control plasmid was named pLVX-Vector. Subsequently, lentiviruses were packaged using 293T cells, and the lentiviruses were used to infect A549 cells. After screening with puromycin, monoclonal cells were obtained from the screened cells using the limiting dilution method, and an A549 cell line stably expressing SNAPIN protein was obtained after expansion culture. The A549 cell line overexpressing SNAPIN was used to infect influenza virus to detect the effect of overexpressing SNAPIN protein on virus replication. Summary of the Invention

[0008] The object of the present invention is to provide a host protein SNAPIN. Through in vitro research, it is found that the SNAPIN protein has the effect of antagonizing influenza virus replication, and the SNAPIN protein has the effect of enhancing the expression of interferon and interferon-induced genes, and has application value in the preparation of anti-influenza virus drugs or immune enhancers.

[0009] To achieve the above object, the present invention adopts the following technical solutions:

[0010] The present invention provides an application of an SNAPIN protein in anti-influenza virus, and the protein is composed of the amino acid sequence shown in SEQ ID NO:1. The sequence of SEQ ID NO:1 is:

[0011] MAGAGSAAVSGAGTPVAGPTGRDLFAEGLLEFLRPAVQQLDSHVHAVRESQVELREQIDNLATELCRINEDQKVALDLDPYVKKLLNARRRVVLVNNILQNAQERLRRLNHSVAKETARRRAMLDSGIYPPGSPGK.

[0012] The protein can have the following applications:

[0013] a. Preparing a drug for inhibiting influenza virus replication;

[0014] b. Preparing an immune enhancer for inhibiting influenza virus replication;

[0015] c. Preparing a drug for inhibiting influenza virus replication in vitro;

[0016] d. Preparing a drug for treating or preventing influenza virus.

[0017] The coding gene of the said protein is a DNA molecule as shown in SEQ ID NO:2.

[0018] The sequence of SEQ ID NO:2 is as follows:

[0019] ATGGCGGGGGCTGGTTCCGCCGCTGTATCGGGGGCAGGGACCCCGGTGGCGGGGCCCACAGGCCGCGACCTTTTCGCCGAAGGGCTGCTGGAGTTCCTGCGACCCGCTGTGCAGCAGCTCGACTCTCACGTACACGCCGTCAGAGAGAGCCAGGTAGAGCTCCGGGAACAAATTGACAACCTAGCCACAGAACTGTGCCGCATAAATGAGGATCAGAAGGTGGCCCTGGATCTTGACCCCTATGTTAAGAAGCTACTTAATGCCCGGCGACGCGTTGTCTTGGTTAACAACATTCTACAGAATGCTCAGGAACGACTGAGACGGCTAAACCACAGTGTTGCCAAGGAAACAGCCCGCAGGAGAGCAATGCTGGATTCGGGAATTTACCCCCCTGGCTCCCCAGGCAAATAA.

[0020] The present invention provides an overexpression vector, an overexpression cell line, an interfering SNAPIN protein expression vector or an interfering SNAPIN gene expression cell line containing the DNA molecule shown in SEQ ID NO:2.

[0021] The present invention constructs a plasmid for knocking down SNAPIN protein using the pLKO.1 vector, named pLKO-SNAPIN. Then, lentivirus is packaged using 293T cells, the lentivirus is infected into A549 cells, and screening is carried out using puromycin. The screened cells are used to obtain monoclonal cells by the limited dilution method, and the A549 cell line with stably knocked-down SNAPIN protein is obtained by expanded culture. The A549 cell line with stably knocked-down SNAPIN protein is infected with influenza virus to detect the effect of knocking down SNAPIN protein on the replication of influenza virus.

[0022] On the A549 cell line with stable overexpression of SNAPIN protein, the qPCR technique is used to detect the expression of type I interferon, type III interferon and interferon-induced genes MxA, OASL, OAS1, IFITM3, ISG56 and ISG15 in the A549 cells with overexpressed SNAPIN protein.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The research of the present invention proves that the SNAPIN protein has the function of inducing interferon expression, stimulating the expression of interferon-induced genes, and playing a role in resisting influenza virus replication, which consolidates the important regulatory function of SNAPIN in influenza virus replication and can be used as an important drug target for anti-influenza virus or as an immune enhancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Results of the effect of overexpressing the SNAPIN protein on inhibiting influenza virus replication; among them Figure 1 A shows the effect of overexpressing the SNAPIN protein on the replication of WSN virus, Figure 1 B shows the effect of overexpressing the SNAPIN protein on the replication of PR8 virus.

[0026] Figure 2 Results of the effect of interfering with the SNAPIN protein on promoting influenza virus replication; among them Figure 2 A shows the effect of interfering with the SNAPIN protein on promoting the replication of influenza virus WSN, Figure 2 B shows the effect of interfering with the SNAPIN protein on promoting the replication of influenza virus PR8.

[0027] Figure 3 Results of the effect of SNAPIN on promoting the expression of interferon and interferon-induced genes (ISGs). DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the present invention claimed, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0029] Materials for implementation: The materials and consumables used in this experimental study can be purchased commercially unless otherwise specified. The 293T cells (human embryonic kidney cell line) were purchased from American Type Culture Collection, ATCC, with the number CRL-3216. The A549 cells (human lung cancer cell line) were purchased from American type culture collection, ATCC, with the number CCL-185. The MDCK cells (dog kidney cell line) were purchased from American Type Culture Collection, ATCC, with the number CCL-34. The plasmid vectors were from Addgene or independently constructed in our laboratory. The influenza virus strains H1N1 (WSN) and PR8 (H1N1) were both preserved in this experiment. The following experiments were all standard molecular biology, cell biology or virology operation methods, which could be easily understood and operated by researchers in this field.

[0030] The following specific examples are used to elaborate the present invention in detail, but do not constitute a limitation to the protection scope of the present invention.

[0031] Example 1. Construction of the plasmid overexpressing SNAPIN

[0032] Extract the RNA of A549 cells using Nucleozol, add oligo(dT) and random primers, and perform reverse transcription to obtain cDNA using HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, R212-01). Use the primers pLVX-SNAPIN-F: CGC GGATCC ATGGCGGGGGCTGGTTCCGCCG and pLVX-SNAPIN-R: CCG GAATTC TTATTTGCCTGGGGAGCCAGGG to amplify the full length of SNAPIN. Digest the SNAPIN fragment and the pLVX-3×Flag vector with BamHI and EcoRl respectively. Recover the digested products by gel electrophoresis, and ligate them using T4 DNA ligase. Transform the ligation product into competent Escherichia coli cells, pick monoclonal colonies, and expand the culture after identifying them as positive. Extract the positive plasmid, name it pLVX-SNAPIN, and send it to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0033] Example 2. Construction of an A549 cell line overexpressing SNAPIN protein

[0034] Seed 293T cells in a six-well plate. When the cell density reaches 80%, use Lipo8000™ transfection reagent (Beyotime, C0533) to transfect the plasmids pSPAX2, PVSV-G, and pLVX-SNAPIN (or pLVX-Vector) into the cells at a ratio of (7.5 μg: 2.5 μg: 10 μg). Replace with fresh medium after 8 h. At 48 h after transfection, collect the supernatant of 293T cells, filter through a 0.22 μm filter membrane to remove cell debris, and obtain lentivirus. Seed A549 cells in a six-well plate, add 2 mL of lentivirus to each well to infect the cells, add Polybrene (final concentration 3 μg / mL) in the dark, centrifuge in a horizontal centrifuge for 2 h, and then place the six-well plate cells in a 37 °C cell culture incubator. After 8 h of infection, discard the supernatant in the six-well plate A549 cells and replace with 10% FBS complete medium. When the six-well plate cells are confluent, passage them into a 10 cm diameter petri dish and add puromycin at a final concentration of 4 μg / mL for screening. Observe cell fluorescence with an inverted fluorescence microscope after 72 h. Continuously add puromycin for screening for 2 weeks until all control cells die. The constructed cell lines are named A549-pLVX-Vector and A549-pLVX-SNAPIN respectively. Detect the overexpression level of SNAPIN using qPCR and Western Blot.

[0035] Example 3. Construction of plasmid interfering with SNAPIN

[0036] Construct a plasmid interfering with the expression of SNAPIN protein using the pLKO.1 vector. The pLKO.1 vector is double digested with AgeI and EcoRI, and the double-digested product is recovered and purified by gel extraction. Design primers pLKO-SNAPIN-F: CCGGCAATGCTGGATTCGGGAATTTCTCGAGAAATTCCCGAATCCAGCATTGTTTTTG and pLKO-SNAPIN-R: AATTCAAAAACAATGCTGGATTCGGGAATTTCTCGAGAAATTCCCGAATCCAGCATTG. According to the instructions of Annealing Buffer for DNA Oligos(5X) (Beyotime, D0251), anneal pLKO-SNAPIN-F and pLKO-SNAPIN-R to form a double-stranded structure. Use T4 DNA ligase to ligate the double-digested pLKO.1 vector and the fragment, then transform Escherichia coli competent cells, pick monoclonal colonies for expansion culture, and name the plasmid pLKO-SNAPIN after plasmid extraction, and send it to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The control plasmid is pLKO-GFP (Addgene, 30323).

[0037] Example 4. Construction of A549 cell line interfering with SNAPIN gene

[0038] Seed 293T cells in a six-well plate. When the cell density reaches 80%, use Lipo8000™ transfection reagent (Beyotime, C0533) to transfect pSPAX2, PVSV-G, and pLKO-SNAPIN (or pLKO-GFP) plasmids into the cells at a ratio of (7.5 μg: 2.5 μg: 10 μg). Replace with fresh medium 8 h later. 48 h after transfection, collect the supernatant of 293T cells, filter through a 0.22 μm filter membrane to remove cell debris, and obtain lentivirus. Seed A549 cells in a six-well plate, add 2 mL of lentivirus to each well to infect the cells, add Polybrene (final concentration 3 μg / mL) in the dark, centrifuge in a horizontal centrifuge for 2 h, and then place the six-well plate cells in a 37 °C cell culture incubator. 8 h after infection, discard the supernatant in the six-well plate A549 cells and replace it with 10% FBS complete medium. When the six-well plate cells grow confluent, passage them into a 10 cm diameter petri dish and add puromycin at a final concentration of 4 μg / mL for screening. Continuously add puromycin for screening for 2 weeks until all control cells die. The constructed cell lines are named A549-pLKO-GFP and A549-pLKO-SNAPIN respectively. Detect the SNAPIN expression level by qPCR and Western Blot.

[0039] Example 5. Overexpression of SNAPIN protein inhibits influenza virus replication

[0040] Use A549-pLVX-Vector and A549-pLVX-SNAPIN cells to seed a 12-well plate. Inoculate the WSN and PR8 strains into the 12-well plate cells at an MOI = 1, incubate at 37 °C for 1 h, and then replace with DMEM maintenance medium containing TPCK (0.125 μg / mL). Harvest the cell supernatant at 12 h, 24 h, 36 h, and 48 h after infection respectively. Use MDCK cells to titrate the virus titer of the harvested cell supernatant, and calculate by the Reed-Muench method. A549 cells overexpressing SNAPIN protein are infected with WSN virus. The virus titer starts to decline at 24 h of detection, and the virus titer drops 562-fold at 48 h ( 50 A). A549 cells overexpressing SNAPIN protein are infected with PR8 virus. The virus titer starts to decline at 36 h of detection, and the virus titer drops the most at 36 h, which is 6.8-fold ( Figure 1 B). The results show that SNAPIN protein can effectively inhibit influenza virus replication. Figure 1 B). The results show that SNAPIN protein can effectively inhibit influenza virus replication.

[0041] Example 6. Interference with SNAPIN protein promotes influenza virus replication

[0042] A549-pLKO-GFP and A549-pLKO-SNAPIN cells were used to seed a 12-well plate. The WSN and PR8 strains were inoculated into the 12-well plate cells at an MOI of 1 and incubated at 37 °C for 1 h. Then, the medium was replaced with DMEM maintenance medium containing TPCK (0.125 μg / mL). The cell supernatants were harvested at 12 h, 24 h, 36 h, and 48 h post-infection. The virus titers of the harvested cell supernatants were titrated using MDCK cells, and the TCID 50 was calculated using the Reed-Muench method. The virus titer of A549 cells with inhibited SNAPIN protein expression increased at 12 h after infection with the WSN virus, and increased 15.8-fold at 36 h ( Figure 2 A). The virus titer of A549 cells with inhibited SNAPIN protein expression began to increase at 36 h after infection with the PR8 virus, and increased 39.5-fold compared with the control after inhibiting SNAPIN protein ( Figure 2 B).

[0043] Example 7. SNAPIN promotes the expression of interferons and interferon-induced genes (ISGs)

[0044] A549-pLVX-Vector and A549-pLVX-SNAPIN cells were used to seed a six-well plate. When the cell density reached 90%, the supernatant was discarded and the cells were collected. Cell RNA was extracted using Nucleozol. cDNA was reverse transcribed according to the instructions of the HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, R212-01). The expression levels of type I interferons (IFN-α, IFN-β), type III interferons (IL-28A and IL-28B), and interferon-induced genes MxA, OASL, OAS1, IFITM3, ISG56, and ISG15 in A549-pLVX-Vector and A549-pLVX-SNAPIN cells were detected by qPCR ( Figure 3 ). The results showed that overexpression of the SNAPIN protein upregulated the expression of IFN-β, IL-28A, IL-28B, MxA, OASL, OAS1, IFITM3, ISG56, and ISG15, indicating that the SNAPIN protein promotes the expression of type I interferons, type III interferons (IL-28A and IL-28B), and ISGs.

[0045] Experiments have proved that SNAPIN protein can inhibit influenza virus replication. After overexpressing SNAPIN protein in A549 cells and then infecting them with influenza virus, the virus titer was significantly reduced compared with the control, indicating that SNAPIN protein can inhibit influenza virus replication at the in vitro cell level. By using the shRNA interference technique, after knocking down SNAPIN in A549 cells and then infecting them with influenza virus, the virus replication can be significantly enhanced. Further, overexpressing SNAPIN protein in A549 cells can promote the expression of type I and type III interferons and interferon-stimulated genes (ISGs). In summary, the present invention identifies a host protein SNAPIN, which has the function of inhibiting influenza virus replication and provides a new option for the preparation and design of anti-influenza virus drugs.

[0046] Obviously, the above embodiments are merely examples for clearly illustrating the present invention and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. Use of a SNAPIN protein in the preparation of a drug against H1N1 influenza virus, characterized in that, The SNAPIN protein is composed of the amino acid sequence shown in SEQ ID NO:

1.

2. The application according to claim 1, wherein The coding gene of the SNAPIN protein is a DNA molecule shown in SEQ ID NO: 2.

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